Crystallographic and Molecular Dynamics Analysis of Loop Motions Unmasking the Peptidoglycan-Binding Site in Stator Protein MotB of Flagellar Motor

Background The C-terminal domain of MotB (MotB-C) shows high sequence similarity to outer membrane protein A and related peptidoglycan (PG)-binding proteins. It is believed to anchor the power-generating MotA/MotB stator unit of the bacterial flagellar motor to the peptidoglycan layer of the cell wall. We previously reported the first crystal structure of this domain and made a puzzling observation that all conserved residues that are thought to be essential for PG recognition are buried and inaccessible in the crystal structure. In this study, we tested a hypothesis that peptidoglycan binding is preceded by, or accompanied by, some structural reorganization that exposes the key conserved residues. Methodology/Principal Findings We determined the structure of a new crystalline form (Form B) of Helicobacter pylori MotB-C. Comparisons with the existing Form A revealed conformational variations in the petal-like loops around the carbohydrate binding site near one end of the β-sheet. These variations are thought to reflect natural flexibility at this site required for insertion into the peptidoglycan mesh. In order to understand the nature of this flexibility we have performed molecular dynamics simulations of the MotB-C dimer. The results are consistent with the crystallographic data and provide evidence that the three loops move in a concerted fashion, exposing conserved MotB residues that have previously been implicated in binding of the peptide moiety of peptidoglycan. Conclusion/Significance Our structural analysis provides a new insight into the mechanism by which MotB inserts into the peptidoglycan mesh, thus anchoring the power-generating complex to the cell wall.


Introduction
The motility protein B (MotB) is a key component of the bacterial flagellar motor. It anchors the MotA/MotB stator ring of the motor to peptidoglycan (PG) of the cell wall and forms part of the proton-conducting channel that couples proton flow to generation of the turning force via an as yet unknown mechanism [1,2]. MotA/MotB units are pre-assembled in the membrane in inactive (closed-channel) form and continuously exchange with the units that form the stator ring [3,4]. Interaction of the MotA/ MotB complex with the flagellar basal body is thought to induce two molecular events: opening of the channel and insertion of the anchor domain of MotB into the PG mesh.
The PG-binding site of MotB resides on the periplasmic Cterminal domain (MotB-C) which shows sequence similarity to outer membrane protein A (OmpA) and related PG-binding proteins [5]. The PG-binding domains are believed to have been acquired by MotBs and other OmpA-like proteins from a common ancestor early in evolution, before MotBs and the outer membrane protein family diverged from each other. The crystal structures of MotB-C from Helicobacter pylori [6] and Salmonella typhimurium [7] revealed that these proteins share a common OmpA-like fold comprising a mixed four-stranded beta-sheet (order 1423), in which three parallel (b1, b2, b3) and one antiparallel (b4) strands are flanked by alpha helices on one side. Although there is no detailed experimental picture of the association between OmpAlike proteins and PG mesh, previous crystallographic and NMR studies on the proteins of this family established the presence of separate recognition sites for glycan and peptide moieties of PG [6,8,9]. Both are located within the three petal-like loops (b1a1 (residues 126-133), b2a2 (163-174) and b3b4 (207-225) in H. pylori MotB) at one end of the b-sheet. Although the PG-binding grooves formed by these loops are topologically very similar in different OmpA-like proteins, all five conserved residues (Gly161, Asp164, Leu179, Arg183, Arg226) that are thought to be essential for PG recognition are buried and inaccessible [6]. This suggests that PG binding is preceded by, or accompanied by, some structural reorganization that exposes the key conserved residues.
Here, we report a new crystal form of H. pylori MotB-C and present the analysis of its structure and dynamics with a Figure 1. The crystal structure of H. pylori MotB-C and the locations of the conserved residues. A: Stereo diagram of the structure of the H. pylori MotB-C monomer. The backbone radius is proportional to the average Ca atom RMSD to the mean structure for the superimposition of the total of 16 monomers in the asymmetric units of Form A and Form B crystals. RMSD values were calculated using Theseus [11] and the figure was prepared using PYMOL [12]. The color gradient runs from blue (the smallest RMSD) to red (the largest RMSD). B: The location of the five residues conserved in the family of OmpA-like PG-binding proteins. The MotB-C monomer is drawn using a ribbon representation. Loops b1a1, b2a2 and b3b4, masking these residues, are colored red. doi:10.1371/journal.pone.0018981.g001 combination of experimental (crystallography) and computational (molecular dynamics simulations) methods. Comparison of the refined structure of this form with the previously reported MotB-C structure [6] provides the evidence for the flexibility of the loops b1a1, b2a2 and b3b4 at the putative PG-binding surface. Principal component analysis (PCA) identifies the concerted opening/closing motions of these loops that are thought to facilitate recognition of MotB by PG by exposing conserved residues in a binding pocket for the peptide moiety of PG.

Overall Structure
The structure of previously unobserved crystal form (Form B) of recombinant H. pylori MotB-C was solved by a molecular replacement approach using AMORE [10]. The coordinates of the previously reported MotB-C structure in a different crystal form (PDB accession code 3CYP [6], hereafter referred to as Form A) were used as a search model. The asymmetric unit of the form B crystal contains 12 subunits. The overall fold of each monomer is very similar to the Form A structure. It contains a mixed fourstranded b-sheet, with three a-helices packing against one face of it, and the fourth forming an N-terminal extension of one of the bstrands ( Fig. 1(A)). The loops connecting b-strands with a-helices are short at one end of the b-sheet (bottom side of the molecule shown in Fig. 1(A)) and long at the other. The three longer petallike loops (b1a1 (residues 126-133), b2a2 (163-174) and b3b4 (207-225)) have been previously implicated in the PG binding by MotB and PG-associated lipoprotein (PAL) [6,9]. PG-binding domains of MotB and PAL share a significant degree of structural similarity [6] and can be functionally interchanged [13] with only partial loss of activity. This strongly suggests that MotB and PAL share a common molecular mechanism of PG recognition. Previous structural studies on MotB and PAL established that loop b2a2 accommodates the binding site for the N-acetylmuramic acid (NAM) moiety of PG [5], whereas the grove between loops b1a1 and b2a2 binds its peptide moiety containing mesodiaminopimelate (m-DAP) [9] (Fig. 1(A)).

Conserved PG-binding residues are buried
Alignment of aminoacid sequences of the C-terminal domains of MotBs from different bacteria identifies eight conserved residues (Fig. S1). Five of them (Gly161, Asp164, Leu179, Arg183, Arg226) are conserved in the entire OmpA family of PG-binding proteins [6] and are therefore likely to be involved in direct binding to PG or in maintaining the fold around residues recognized by PG. Two residues in particular, Asp164 and Leu179, play a critical role in recognition of the peptide moity of PG. A previous NMR study on the complex between Haemophilus influenzae PAL and a synthetic PG precursor [9] demonstrated that the PAL residues Asp71 and Leu82, equivalent to Asp164 and Leu179 in H. pylori MotB, form contacts with the m-DAP residue of a synthetic PG precursor, involving a hydrogen bond to the side chain of Asp71 and a hydrophobic interaction with the side chain of Leu82. Structural analysis and calculations of the accessible side-chain surface area in Form A and Form B crystals of H. pylori MotB-C and in the crystal structure of the periplasmic domain of Salmonella MotB [7] ( Table 1) show that all five conserved residues are clustered on one side of the molecule and are completely shielded from the solvent by loops (b1a1), (b2a2) and (b3b4) ( Fig. 1(B)). Therefore, we hypothesize that PG binding is preceded by or accompanied by some conformational transition in MotB that exposes the key conserved residues.

Conformational variability of the conserved loops at the PG binding site
One well recognized approach to obtain information about conformational movements in proteins is comparison of structures of independent monomers in the crystallographic asymmetric unit or comparative analysis of different crystal forms of the same molecule [14][15][16]. To compare the structures of the individual monomers in the asymmetric units of the Form A and Form B crystals, the monomer structures were superimposed and analysed for Ca atom root-mean-square-deviation (RMSD) to establish regions of conformational variability. The most significant differences are observed in the structures of loops b1a1, b2a2 and b3b4 at the PG binding site ( Fig. 1(A), 2(A)), with the following residues showing the largest displacements of Ca atoms: Glu126 (2.3 Å ), Asn127 (2.4 Å ) (loop b1a1), Val169 (2.8 Å ), Lys170 (4.1 Å ) (loop b2a2) and Asp216 (2.4 Å ) (loop b3b4). Analysis of the distribution of the main-chain temperature factor averaged over the total of 12 monomers in the asymmetric units of the Form B crystals also highlights these three loops as the most variable regions in the structure (Fig. 2(B)). Furthermore, the monomer structure superimpositions and the temperature factor analysis over the equivalent residue range in the three available crystal forms of S. typhimurium MotB-C [7] (Fig. 2) identifies the loops equivalent to loops b2a2 and b3b4 in H. pylori protein as highly mobile thus supporting the hypothesis that the loop movements at the PG-binding site are a universal feature of MotB proteins.
Structure superimposition shows that there are significant differences in conformations of many side chains and the orientation of the main-chain peptide groups in loops b1a1, b2a2 and b3b4. These differences are linked to the differences in the networks of hydrogen bonds that stabilize the loop structure. For instance, in one of the conformations, the side chain of Asp127 forms a hydrogen bond with the main-chain peptide of Ala128, whereas in a different conformation, it is hydrogen-bonded to the main-chain NH-group of Thr129. Reorientation of the main-chain peptide groups leads to formation/ breakage of the hydrogen bonds Ala128(O) -Arg-183(Ne), Leu168(O) -Thr171(N), Leu168(O) -Thr171(Ob), Asn215(O) -Arg221(Ne), Asp216(O) -Arg221(Nf) and Asp216(Oc) -Asn220(Nd). All the loop residues that show side-chain conformational variability in the analysed crystal structures have been previously implicated in PG binding. Residues 126-129 form a stretch that is structurally equivalent to H. influenzae PAL residues Gly35, Phe36 and Asp37, the latter two being involved in binding to the peptide moiety of a PG precursor [9]. Residues 168-171 and 215-216 belong to loops b2a2 and b3b4 which in turn, have been implicated in recognition of the glycan chain of PG [6]. The length and the sequence of the long loops show significantly less cross-species variation than those of the short loops (a1b2, a2b3 and b4a4) at the opposite end of the b-sheet (Fig.  S1). Thus, our crystallographic analysis suggests that the flexibility of the semi-conserved loop region b1a1, b2a2 and b3b4 is important for PG-binding activity of MotB. The biological function of proteins requires the ability to change conformation [17]. In particular, the structural flexibility has been associated with molecular recognition [18]. Flexibility around the ligand-binding site improves its accessibility, facilitates the ligand entry and allows subsequent optimisation of the surface complementarity to maximise the number of contacts between ligand and protein upon binding. The variations of MotB petal-like loops b1a1, b2a2 and b3b4 identified through our crystallographic analysis are thought to reflect natural flexibility at this site required for insertion into the PG mesh. Indeed, it has been previously established that the size of the MotB-C dimer is very close to the size of the pore in the PG mesh (approximately 70 Å ) [6,19]. Therefore, the PG-binding surface on MotB-C must be flexible enough to allow its insertion into the PG pore. This hypothesis can be tested by introducing rigidity into the PG-binding loops (via proline substitutions or engineered disulfide bridges) and then testing the activity of the resultant MotB mutants in vivo. A useful insight into conformational rearrangements in MotB required for peptidoglycan (PG) binding can also be gained through determination of the structure of MotB-C complex with a peptidoglycan fragment.

Role of the concerted motions of the petal-like loops in MotB function
An important functional aspect of the loop flexibility at the PG binding site lies in their ability to mask/unmask the five conserved residues critical for PG recognition. In order to understand how concerted motions of these loops may expose the cluster of the buried conserved residues (Fig. 1(B)), we performed mass-weighted principal component analysis (PCA) of the molecular dynamics (MD) simulation of the MotB-C dimer. We chose to perform PCA of MD simulations rather than crystal structures to avoid bias imposed by crystal contacts. For validation, theoretical B-factors have been calculated for the MD trajectory and showed a very similar trend to the crystallographic B-values (Fig. 2(B)). PCA produces so called principal modes revealing the concerted motions within a molecule and their directions. Typically, the first few principal modes explain most of the motion observed during the MD simulation [20]. Here we describe the loop motions in two chains comprising the dimer along the first three eigenvectors, which account for 44% and 42% of the overall motion of chains D and E respectively.
Chain D mode 1 (motion profile 1 (Fig. 3(A))) accounts for 24% of the overall motion of this chain. In this mode, part of loop b1a1 moves in concerted fashion with loop b3b4 and in an opposite direction to loop b2a2 and residues 126-128 in loop b1a1. Upon this movement, the grooves harbouring conserved MotB residues (including Asp164 and Leu179 implicated in binding to the peptide moiety of PG) open up making them accessible to PG. Motion profile 2 (Chain E mode 2, Fig. 3(B)) accounts for 13% of the overall motion and is dominated by the movements of loops b2a2 and b3b4 resulting in the opening of the cleft between these two loops. This could increase accessibility of Asp164, and Leu 179 to a lower extent. The motions of the petal-like loops in modes 2 and 3 from chain D (accounting for 20% of the overall motion) and modes 1 and 3 from chain E (accounting for 29% of the overall motion) show only minor differences in their extent and exact directions and are therefore grouped into a single motion profile 3 (Fig. 3(C)). In this motion profile, loops b2a2 and b1a1 move concomitantly in nearly opposite direction to loop b3b4. This movement is likely to promote cleft widening between b3b4 and b2a2, exposing Asp164.
Thus, the results of the MD simulations are consistent with and also extend the crystallographic analysis, and provide evidence that the three loops move in a concerted fashion, likely to expose conserved MotB residues that have previously been implicated in binding of the peptide moiety of PG. The intrinsic conformational variability of the surface-exposed PG-binding residues and the evidence of intramolecular motions promoting exposure of the buried ones suggest that PG recognition by OmpA-like proteins may occurs via a conformational selection rather than an induced fit mechanism.

Materials and Methods
Protein production, crystallization and data collection Recombinant H. pylori MotB-C was expressed in E. coli and purified as described previously [8]. Protein was concentrated to 8 mg/ml (based on the Bradford assay [21]) and centrifuged for 20 min at 13,000 g to clarify the solution. The crystals have been obtained by the sitting-drop vapour-diffusion method using the drops containing 3 ml of the protein solution mixed with 3 ml of the reservoir solution of 15% PEG 3350 and 200 mM sodium tartrate, and equilibrated against 500 ml of the reservoir solution at 293 K. For data collection, crystals were flash-cooled to 100 K after soaking in a cryoprotectant solution containing 22% PEG 3350, 200 mM sodium tartrate and 20% (v/v) glycerol. X-ray diffraction data were collected to 2.5 Å resolution using the Swiss Light Source (PX06, Villigen, Switzerland), and processed and scaled using programs MOSFLM [22] and SCALA [23] (see Table S1). These crystals belong to space group P2 1 with unit-cell parameters a = 107.6, b = 100.3, c = 108.5 Å , b = 119.5u.

Structure Determination and Analysis
The structure of the new crystal form of MotB-C was solved by molecular replacement using AMORE [10] with a crystallographic P222 tetramer observed in the previously reported form (RCSB PDB code 3CYP [6]), as a search model. The asymmetric unit contains three tetramers related by a pseudo three-fold symmetry about the axis approximately parallel to b. Model building and refinement were carried out using programs COOT [24] and REFMAC (CCP4 [23]), without non-crystallographic symmetry restraints. The REFMAC TLS refinement option was employed, with each individual monomer treated as one entity. The final protein model was validated using MolProbity [25]. Final refinement statistics is summarized in Table S1.
Structure superpositions were performed using the program Theseus [11]. Sequence alignment was carried out using the software CLUSTALW2 (http://www.ebi.ac.uk/Tools/clustalw2/ index.html). Accessible surface area was calculated using Areaimol in CCP4 [23] with a probe radius of 1.4 Å .

Molecular dynamics (MD) simulations and PCA analysis
MD simulations were performed on the MotB-C dimer comprising chains D and E of the high-resolution crystal structure PDB 3CYP [6] using NAMD2 (version 2.7b1) [26] and the CHARMM27 force field with CMAP correction [27]. Missing hydrogen atoms were added using VMD [28]. The protein was then solvated into a 86 Å 686 Å 686 Å box of water (TIP3 model). This resulted in a chargeless system of 59,822 atoms. After minimisation and equilibration of the water and ions with the protein fixed the system was equilibrated in an NPT ensemble (P = 1.0 bar; T = 310 K) for 1 ns where positional constraints on the protein were smoothly relaxed. The simulation was run for 126.5 ns under the same conditions with periodic boundary conditions and a time step of 2 fs. Van der Waals interaction cutoff was set to 12 Å and long-range electrostatic forces were computed using the particle-mesh Ewald summation method. Mass-weighted PCA (or quasiharmonic analysis) [29] of the MD trajectory was used to determine the principal components of motion for each MotB-C monomer within the dimer (5 ps frame rate). Massweighted covariance matrices of each monomer were diagonalised and the principal modes extracted using ptraj (AmberTools) [30]. The first three modes were then individually projected onto the Cartesian space.

Supporting Information
Table S1 X-ray data collection and refinement statistics. (DOC)